LOSS OF INTRACELLULAR PUTRESCINE POOL-SIZE REGULATION INDUCES APOPTOSIS

Synthesis and uptake are two important regulated mechanisms by which e ukaryotic cells maintain poly amine levels. The role that loss of synt hesis and/or uptake regulation plays in mediating putrescine toxicity was investigated by comparing toxicity in an ornithine decarboxylase ( ODC)-deficient Chinese hamster ovary cell line (C55.7) with a function al putrescine transport system and an ODC-overproducing rat hepatoma c ell line (DH23b), which are transport regulation deficient. When C55.7 cells were transfected with either mouse ODC (M) or trypanosome ODC ( Tb), intracellular putrescine content increased slightly in C55.7(Tb-O DC), compared to C55.7(M-ODC), due to the lack of response of Tb-ODC t o polyamine regulation. The increase in putrescine content resulting f rom loss of ODC regulation had no impact on cell growth and viability. When the feedback repression of polyamine uptake was blocked with cyc loheximide, C55.7 cells transfected with either ODC construct accumula ted very high levels of putrescine from the medium, and underwent apop tosis in a putrescine dose-dependent manner. A similar correlation of deregulated putrescine uptake and increased apoptotic cells was observ ed in DH23b cells. These data demonstrate that loss of feedback regula tion on the poly amine transport system, but not ODC activity, is suff icient to induce apoptosis. Thus, downregulation of the transport syst em is necessary to prevent accumulation of cytotoxic putrescine levels in rodent cells. (C) 1997 Academic Press.